IL-15 is a major factor regulating the development and homeostasis of CD8 T cells and NK cells as well as their responses during immune stimulation. During the steady state, IL-15 responses are mediated via transpresentation, where cell surface IL-15, associated with IL-15Ra, stimulates neighboring cells during a cell- cell interaction. Conversely, studies have found that soluble (s) IL-15Ra/IL-15 complexes, with potent stimulatory activity, are produced by Toll-like receptor stimulation and after lymphodepletion in humans and mice. Additionally, we have recently shown that sIL-15Ra/IL-15 complexes are generated in response to viral infection, Interferon-a, and CD40 stimulation. Since cell surface IL-15Ra/IL-15 also increases under these conditions, the role of sIL-15Ra/IL-15 complexes is presently unclear. Because generation of sIL-15Ra/IL-15 complexes coincides with enhanced IL-15 responses, we hypothesize that sIL-15Ra/IL-15 complexes enhance lymphocyte responses during immune stimulation. Our long-term goals are to elucidate the role of sIL-15Ra/IL- 15 complexes generated in response to pathogen infections, vaccination, autoimmune diseases, and cancer so that we can identify novel ways to regulate CD8 T cells and NK cells. Previous studies suggest IL-15Ra/IL- 15 complexes are cleaved from the surface by the metalloproteinase ADAM17. Moreover, the cleavage site in the IL-15Ra and a critical amino acid required for cleavage has been identified. Therefore, the goal of these studies is to use this knowledge to generate transgenic (Tg) mice expressing a cleavage-resistant IL-15Ra that prevents production of sIL-15 complexes but leaves IL-15 transpresentation intact. This model system can then be used to distinguish the immunological roles of endogenously-produced soluble IL-15 complexes from those mediated by IL-15 transpresentation. To accomplish this goal, we propose the following two specific aims: 1. Develop genetically engineered mice expressing cleavage-resistant IL-15Ra. Constructs encoding cleavage-resistant IL-15Ra or Wt IL-15Ra driven by a MHC class I promoter will be inserted into IL- 15Ra-/- blastocytes for generation of Tg mice. Mice expressing the transgene at the DNA and protein level will be identified and bred to the IL-15Ra-/- background. 2. Determine effect of transgenic cleavage-resistant IL-15Ra on development of IL-15 responsive lymphocytes and expression of sIL-15 complexes. The general phenotype of T cells and NK cells will be examined in untreated cleavage-resistant IL-15Ra Tg+ and Wt IL-15Ra Tg mice to measure the restoration of IL-15 function in lymphocyte development and homeostasis. Expression of serum sIL-15Ra/IL-15 complexes will be measured in Tg+ mice after viral infection or other types of immune stimulation. Developing this model will allow us to identify the importance of sIL-15Ra/IL-15 complexes generated by various types of immune stimulation. The knowledge obtained will provide the critical insight needed to develop therapeutic strategies to promote IL-15 responses that can enhance immune responses to tumors or pathogens as well as block IL-15 responses that promote inflammatory events.